This invention relates generally to enzyme extraction and purification and, more particularly, to the extraction and purification of plasminogen activating enzymes by affinity chromatography.
Plasminogen activating enzymes, such as urokinase, cytokinase, and the like, catalyze the conversion of plasminogen to plasmin, an enzyme which is capable of lysing fibrin clots. The plasminogen activating enzymes have been shown to have therapeutic value when injected in humans as an effective thrombolytic agent for dissolving blood clots. Various biological sources are known to contain plasminogen activating enzymes in low concentrations. For example, urokinase is a plasminogen activating enzyme present in mammalian urine, and cytokinase is a plasminogen activating enzyme present in mammalian body tissue. Other plasminogen activating enzymes are present in plasma and in spent tissue culture growth medium.
Various attempts have previously been made to extract and purify the plasminogen activating enzymes from their biological sources. These attempts have depended for the most part on inefficient, expensive and time-consuming multi-step procedures involving a combination of several different extraction and purification techniques, including precipitation, centrifugation, dialysis, ion exchange chromatography and gel filtration chromatography.
The potentially simpler and more efficient extraction and purification technique of affinity chromatography, which has been used in recent years in the extraction and purification of some other types of enzymes, has not met with any appreciable success when applied to the extraction and purification of plasminogen activating enzymes. In affinity chromatography of an enzyme, a biospecific extracting agent is employed which comprises an insoluble solid support material having covalently coupled to its surface an affinity ligand having biospecificity for the particular enzyme, so that the enzyme becomes preferentially adsorbed or bound to the extracting agent by specific interaction of the enzyme with the ligand. The affinity ligand is typically a competitive inhibitor for the particular enzyme, i.e., a material which interacts with the enzyme to form a complex therewith and temporarily inhibit the enzymatic activity thereof. The principle drawback up to now to the successful use of affinity chromatography techniques in the extraction and purification of plasminogen activating enzymes from their biological sources has been the fact that the previously known competitive inhibitors for plasminogen activating enzymes have all been relatively weak competitive inhibitors lacking sufficient affinity and specificity to the plasminogen activating enzymes to make them suitable for use as biospecific affinity chromatography ligands.